Wound healing in humans and other mammals is often inadequate and incomplete. Delayed healing markedly increases hospitalization costs, and often the wound continues as a chronic sore that requires extensive attention and medical care in order to control infection and tissue necrosis. Even when such wounds finally heal, the "wound area" is often devoid of the ability to respond to tactile stimulus, and is often filled with excessive deposits of collagen that lead to permanent scarring. The need for improved wound healing also extends to wounds generated by surgical procedures. For instance, although cosmetic surgery is one of the fastest growing medical specialty areas, the success of such procedures is limited by the adequacy of healing in the typically adult and elderly clientele. Further, hair transplants often fail due to an inadequate blood supply around the transplant. Enhanced healing and neovascularization of the transplant would enhance the establishment of the graft.
The rapidity of reestablishment of a biological coverage on wound surfaces is a critical element in the healing prognosis. Natural open wounds are first covered by a blood and plasma exudate which dries to form the initial "scab" that covers the wound. This scabby layer forms a short-term protective coverage from outside elements while healing proceeds under this layer.
For longer-term coverage of extensive wounds, surgeons often resort to transplants in which a thin piece of superficial skin (called a "split-thickness skin graft") is implanted over the wound to form an island of skin cells that can overgrow the surface. Deeper skin wounds often require a more extensive skin transplant (called a "full-thickness skin flap") in which the entire skin down to the muscular layers is moved to cover the wounds. Split-thickness flaps are hampered by the low degree of surgical "take." Typically, only about 20% to 40% of the transplanted skin successfully reestablishes itself in its new position. Full-thickness flaps are even more difficult to reestablish in a new site. Surgeons are usually constrained to leave one end of the flap attached to a blood supply, while the other end is stretched to the new site to be sewn in place. Only after the transplanted end of the flap reestablishes a new blood supply is the other end of the flap moved to the new site to complete the transplant. Such procedures often result in extensive loss of tissue and additional pain and suffering for the patient.
Wound healing can be divided into four essential components: inflammation, angiogenesis, collagen deposition and epithelialization. All of these play a role in the healing of all wounds.
In recent years, a number of protein factors have been shown or implicated to be useful in wound healing. These factors are essential to the growth and differentiation of the cells which serve to replace the tissue destroyed. A number of candidate factors have been identified on the basis of the ability of extracts from various tissues, such as brain, platelets, pituitary, and hypothalamus, to stimulate the mitosis of cultured cell lines. These factors include transforming growth factors (TGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), insulin-like growth factors (IGF), epidermal growth factor (EGF), and a myriad of other proteins. This invention shows that thrombospondin has now been identified as an alternative protein which may be used in a similar fashion to promote wound healing.
Thrombospondin (also known as thrombin sensitive protein or TSP) is a large molecular weight 180 kD glycoprotein composed of three identical disulfide-linked polypeptide chains. TSP is stored in the alpha-granules of platelets and secreted by a variety of mesenchymal and epithelial cells (Majack et al., Cell Membrane (1987)3:57-77). Platelets secrete TSP when activated in the blood by such physiological agonists such as thrombin. TSP has lectin properties and has a broad function in the regulation of fibrinolysis and as a component of the extracellular matrix (ECM). TSP is one of a group of ECM proteins which have adhesive properties. Other ECM proteins include laminin, fibronectin and fibrinogen. TSP binds to fibronectin and fibrinogen (Lahav, et al., Eur. J. Biochem. (1984) 145:151-6) and these proteins are known to be involved in platelet adhesion to substratum and platelet aggregation (Leung, J. Clin. Invest (1986) 74:1764-1772).
Lawler (J. Biol. Chem. (1978) 253:8609-16) first described the purification of TSP from the alpha granules of activated platelets using exclusion chromatography. TSP has subsequently been purified by heparin affinity chromatography (Lawler et al., Thromb Res (1981) 22:267-269), fibrinogen affinity chromatography (Tuszynski et al., J. Biol. Chem. (1985) 260:12240-5), barium chloride precipitation (Alexander et al., Biochem. J. (1984) 217:67-71) and anion exchange chromatography with HPLC (Clezardlin et al., J. Chromatog. (1984) 296:249-56).
The complete amino acid sequence of TSP has been deduced from DNA clones prepared by various groups including Lawler et al., J. Cell Biol. (1986) 103:1635-48, Kobayashi et al., Biochemistry (1986) 25:8418-25, Dixit et al., Proc. Ntl. Acad. Sci. (1986) 83:5449-53 and Hennessy et al., J. Cell Biol. (1989)108:729-36.
Work from several laboratories has implicated TSP in response of cells to growth factors. Submitogenic doses of PDGF induce a rapid but transitory, increase in TSP synthesis and secretion by rat aortic smooth muscle cells. (Majack et al., J. Biol. Chem. (1985) 101:1059-70). PDGF responsiveness to TSP synthesis in glial cells has also been shown. (Asch et al., Proc. Ntl. Acad. Sci. (1986) 83:2904-8). TSP mRNA levels rise rapidly in response to PDGF (Majack et al., J. Biol. Chem. (1987) 262:8821-5). TSP acts synergistically with epidermal growth factor to increase DNA synthesis in smooth muscle cells (Majack et al., Proc. Ntl Acad Sci (1986) 83:9050-4) and monoclonal antibodies to TSP inhibit smooth muscle cell proliferation (Majack et al., J. Biol Chem (1988) 106:415-22). TSP modulates local adhesions in endothelial cells.
The TSP protein sequence includes the X-RGD-Y sequence first described by Ruoslahti (U.S. Pat. No. 4,578,079, U.S. Pat. No. 4,614,517 and U.S. Pat. No. 4,792,525). Ruoslahti discloses that the RGD sequence is believed to confer adhesive properties. However, other distinct non-RGD peptides have been identified in proteins which contain the RGD sequence elsewhere, and those non-RGD peptides have also been shown to confer adhesive properties (Tashiro et al., J. Biol. Chem. (1989) 264:16176-82).
Varani et al. (J. Clin. Invest. (1988) 81:1537-1544) shows that TSP has an effect on the differentiation of human epidermal keratinocytes and suggests that TSP may participate in reepithelialization during wound repair.
Immunostaining studies have indicated that TSP may be present in the extracellular matrix of wounds (Raugi et al., J. Invest. Dermatol. (1987) 39:551-554). However, the mere presence of TSP in a wound does not demonstrate its use in healing wounds.
The use of TSP or its derivatives has never before been shown to improve wound healing or to increase the rate at which wounds heal.